While GABA is the main inhibitory transmitter in the brain, the last three decades have seen an increased appreciation of the inhibitory role played in the brain by another amino acid, glycine (Gly) (Lynch, 2009). Gly has long been known to dominate inhibition via strychnine-sensitive Gly receptors (GlyRs) in the lower CNS centres, for example spinal cord where its analgesic actions are well documented (San Martin et al., 2022, Zeilhofer et al., 2021).

As members of the ligand-gated ion channel superfamily, GABAARs and GlyRs share structural similarity. Thus, it is unsurprising that a large body of evidence has documented modulation of GlyRs by ethanol (Burgos et al., 2015, Perkins et al., 2010, Soderpalm et al., 2017). In common with GABAARs, strychnine-sensitive GlyRs exhibit a pentameric structural assembly with the five composing subunits being drawn from a more restricted repertoire cf the GABAAR, that is α1–4 and β. Also, functional GlyRs can be assembled as homomeric α subunit composed, or heteromeric complexes, containing both α and β subunits (Burgos et al., 2016, Lynch et al., 2023).

Ethanol potentiates both native and recombinant GlyRs, albeit receptor-subtype and neuron selective differences are apparent (Burgos et al., 2015, Harrison et al., 2017, Lovinger and Roberto, 2023, Soderpalm et al., 2017). One interesting feature of ethanol potentiation of GlyR cf GABAAR function (Belelli et al., 2021, Korpi et al., 2007) is that it has been reported consistently to occur at concentrations relevant to human consumption that is 10–100 mM (Harrison et al., 2017), and especially in the lower concentration range that is 10–30 mM. Detailed studies in recombinant GlyRs indicate a rank order of selectivity of ethanol for homomeric α1- versus α2-GlyRs (Mascia et al., 1996, Yevenes et al., 2010). However, inclusion of the β subunit appears to modify the pharmacology, as α2β–GlyRs show greater potentiation by low concentrations of ethanol, that is 10 mM (Munoz et al., 2021). Also, investigations of ethanol action upon native GlyRs reveal apparent region- and neuron- selective effects, which are consistent with the reported subunit selectivity in recombinant receptor studies for example increased potentiation at α1 GlyR -rich neuronal population such as Medium Spiny Neurons of the NAc (Araya et al., 2022, Forstera et al., 2017). Moreover, potentiation of GlyR-mediated synaptic and extrasynaptic inhibition occurs in anatomical sites relevant to ethanol behavioural actions (Harrison et al., 2017, McCracken et al., 2017) for example dorsal raphe (DR) (Maguire et al., 2014), lateral habenula (LHab) (Li et al., 2019), cortex (Badanich et al., 2013, Salling and Harrison, 2014), Ventral Tegmental Area (VTA) (Araya et al., 2021) and NAc (Forstera et al., 2017), reviewed in Lovinger & Roberto, 2023.

As elaborated further below, GlyR may be targeted to reduce alcohol harms via a two-pronged approach, that is (1) to limit alcohol preference and intake and (2) to counteract the anxiety that accompanies and escalates chronic alcohol consumption. Evidence in support of both follows. In common with GABAAR-based strategies, a key question in an approach to reduce alcohol harm concerns the dissection of the repertoire of ethanol-mediated behavioural effects that may be mediated or contributed by an action on GlyRs. As for GABAAR, the identification of the molecular determinants of EtOH effects on GlyRs has proved instrumental in this respect. Thus, using mice incorporating α1-GlyR with blunted ethanol sensitivity, the α1-GlyR subunit has been implicated in the sedative and locomotor stimulant actions of ethanol (Aguayo et al., 2014, Blednov et al., 2012, McCracken et al., 2013). In addition, α1-GlyRs have been implicated, albeit not consistently, in ethanol consumption and preference. Thus, for example, knock-in (KI) mice where a reduction in ethanol sensitivity is engineered in the α1 subunit, exhibit reduced ethanol consumption and preference (McCracken et al., 2013, Munoz et al., 2020). Also, in the NAc GlyRs have been documented to play a protective role in the regulation of dopamine release to lower ethanol consumption (Molander and Soderpalm, 2005, Molander et al., 2007). This action appears to be partially mediated by α1GlyRs (Araya et al., 2022). These reports suggest that region- and neuron-selective α1-GlyR-mediated effects are important in EtOH regulation of the reward circuit. However, the concomitant expression of other inhibitory signals for example other GlyR besides α1-GlyRs and/or specific GABAAR subtypes in selective locations of the neuronal circuitry may also contribute. This proposal is consistent with the observation that α1-GlyRs are indeed expressed in various arms of reward circuit for example NAc (Munoz, Yevenes, Forstera, Lovinger, & Aguayo, 2018), and VTA (Araya et al., 2021). However, a concomitant and higher expression of both α2 and α3-GlyRs has also been reported for the NAc (Ceder et al., 2023, McCracken et al., 2017). Additional support derives from genetic Knock-Out (KO) studies implicating both specific GABAAR subtypes (Stephens, King, Lambert, Belelli, & Duka, 2017) and α3-GlyRs in alcohol addiction (Han, Gelernter, Kranzler, & Yang, 2013). In further agreement, mice null for α2-GlyRs (α2 KO) exhibit reduced, while α3-GlyRs KO increased ethanol intake and preference in a 24 h two-bottle test. However, crucially, anxiolysis, motor incoordination, loss of righting reflex, and acoustic startle response, are not altered in these mutants (Blednov et al., 2015). Collectively, these reports suggest that the documented protective role of GlyRs in the NAc may be exploited therapeutically for example via α1 and, possibly α2-GlyRs to manage alcohol consumption and reduce its harm. The specific mechanisms whereby GlyRs regulate DA release in the NAc remain to be fully understood although an important role for the ethanol-sensitive α1- GlyR in the VTA (Ye et al., 2004) has been suggested (Soderpalm et al., 2017).

Enhancing endogenous levels of glycine may also be a potential therapeutically viable route to reduce chronic alcohol intake in humans. Interestingly, systemic administration of selective inhibitors of glycine transporter (GlyT) 1, ORG25935 and 24598 both increase Gly and reduce EthOH consumption and preference in rodents (Lido et al., 2009, Lido et al., 2012, Lido et al., 2017, Molander et al., 2007, Vengeliene et al., 2010). However, a short, one-dose, clinical trial in a population of alcoholics failed to prevent relapse and reproduce the GlyT-inhibitors blocking effects on EtOH consumption in humans (De Bejczy et al., 2014). Future better designed and powered clinical studies may provide more conclusive answers as to the therapeutic potential of GlyT1 inhibitors in the management of chronic alcohol intake.

Besides reducing alcohol consumption via an action upon key centres within the reward circuits, there is a second reason whereby GlyRs may prove an effective target to reduce alcohol harms. This relates to the role of GlyRs in the regulation of negative emotions that accompany chronic alcohol consumption. Anxiety and depression are important and integral components of alcohol dependence (Anker and Kushner, 2019, Kushner et al., 2012) contributing both to drinking initiation and to chronic alcohol consumption (Koob, 2003). When considering the role of GABAARs in the anxiolytic actions of ethanol, despite the similarity of the anxiolytic actions of BZs and ethanol, α2(S270H/L277A) KI mice, engineered to express ethanol-insensitive α2-containing GABAAR receptors retain ethanol anxiolytic properties although do not develop conditioned taste aversion to ethanol and exhibit a decreased ethanol-induced hypnosis together with a complete loss of ethanol-induced motor stimulation (Blednov et al., 2011). These findings suggest that molecular targets other than GABAARs may be implicated in the anxiolytic actions of ethanol. Thus, the potential role played by and the specificity and anatomical basis of GlyRs in the anxiolytic actions of EtOH is of interest.

Amongst the anatomical substrates implicated in the negative emotions associated with chronic alcohol consumption, the LHab has received significant attention. Specifically, an increase in both glutamatergic transmission and neuronal activity of LHab neurons is documented to accompany the aversive behaviour associated with and putatively driving chronic alcohol consumption (Gregor et al., 2019, Shah et al., 2017). In support, block of glutamatergic, AMPA-mediated transmission (Li et al., 2017) or normalization of GLU transport (Kang, Li, Bekker, & Ye, 2018) or, more generally, restoration of neuronal activity via inhibition of the M current (Kang et al., 2017), alleviates depressive and anxiety behaviour respectively, and reduces EtOH intake in rats. Of related relevance, interestingly, a recent study has revealed an important and novel role for a tonic GlyR conductance in the regulation of LHab neuronal excitability. What is more, remarkably, is that activation of the strychnine-sensitive GlyR in the LHab reverses the anxious phenotype that accompanies chronic alcohol intake (Li et al., 2019). The anti-anxiety profile associated with GlyR activation presumably reflects normalization of LHab neuronal activity, which is known to drive downstream inhibition of the monoaminergic (dopaminergic, serotonergic, and noradrenergic) neuronal ensembles via GABAergic rostromedial tegmental (RMTg) neurons or midbrain local GABAergic interneurons (Ables et al., 2023, Metzger et al., 2021).

Besides the lateral habenula, GlyRs are expressed in another behaviourally relevant anatomical locus, namely the DR, also important for the anxiolytic effects of EtOH (Fu et al., 2017, LeMarquand et al., 1994a, LeMarquand et al., 1994b, Pistis et al., 1997). Thus, a large glycinergic conductance has been described for DR neurones, and moreover, this conductance is greatly potentiated by EtOH 30 mM (Maguire et al., 2014). The robust enhancement of the inhibitory, presumed α1-mediated glycinergic conductance by 30 mM EtOH, a relatively low concentration, is most likely behaviourally important. Specifically, the greatly increased GlyR-mediated inhibition is predicted to block the downstream 5-HT2C receptor-mediated excitation of a CRH microcircuit, implicated recently in the inhibition of the GABAergic anxiolytic output of the Bed Nucleus of the Stria terminalis to the VTA and lateral hypothalamus (Marcinkiewcz et al., 2016).

Collectively, these findings add to a growing appreciation of the previously neglected role of glycinergic, mainly extra synaptic-mediated, inhibition in neuronal brain networks relevant to ethanol actions and suggest that development of GlyR-based therapeutics, may offer an alternative route to both effect anxiolysis and reduce ethanol consumption and, hence alcohol harms. In indirect support of the putative therapeutic value of this approach, a drug already used to treat alcohol withdrawal for example chlormethiazole at clinically relevant concentrations greatly enhances both GlyR function in spinal cord neurones (Hales & Lambert, 1992) and the glycinergic tonic conductance in DR slices (Maguire et al., 2014).